1. Field of the Invention
The present invention is generally directed to the use of X-ray contrast media to block antigen-antibody complexes. The present invention is more specifically directed to the use of contrast media as drugs to inhibit allergic reactions, to treat or prevent allergic conjunctivitis, allergic rhinitis, their use in “rush” and “routine” immunotherapy and in non-responding anaphylaxis.
2. Description of the Related Art
It has been known for many years that individual X-ray contrast media (“CM”) have a varying potential to produce reactions that resemble in every respect the anaphylactic reactions that occur in some individuals subjected to antigens to which they have a known hypersensitivity (Shehadi, W. H., AJR 1975, 124, 145-152; Lasser, E. C. et al., Radiology 1997, 203, 605-610; Katayama, H. et al., Radiology 1990, 175, 621-628). X-ray contrast media reactions, however, occur in individuals regardless of previous exposure, and no one has been able to reliably demonstrate the presence of specific antibodies in these patients or in experimental animals injected with any of the contrast media unless these media were artificially bound to a protein prior to injection (Carr, D. H. and Walker, A. C., Br. J. Radiology 1984, 57, 469-473; Brasch, R. et al., Invest Radiology 1976, 2, 1-9; Lasser, E. C. et al., AJR 1962, 87, 338-360; Dunn, C. R., Lasser, E. C. et al., Invest Radiology 1975, 10, 317-322). While previous exposure to contrast media is not necessary for a patient to develop a reaction, reactions occur more commonly in individuals with a history of an allergy of any sort (Katayama, H. et al., Radiology 1990, 175, 621-628; Lasser, E. C. et al., Radiology 1997, 203, 605-610). Most (non-contrast media) clinical allergic reactions occur when a person is exposed to an antigen having the molecular properties of a protein or of a smaller molecule that can be shown to bind to a protein in vitro (a “hapten”). X-ray contrast media have neither of these attributes. Some out-of-date media demonstrated a degree of binding to some serum proteins, but this was never sufficient to allow the media to act as haptens (Lasser, E. C. et al., AJR 1962, 87:338-360). In light of these considerations, the reactions that sometimes occurred after contrast media injections were termed “anaphylactoid” rather than true “anaphylaxis.” True “anaphylaxis” is known to require the release of histamine and other mediators present within either mast cells or basophilic cells. The former can be found within tissues while the latter are present in the blood stream. While it could be demonstrated that histamine release occurs in X-ray contrast media reactions (Lasser, E. C., Walters, A. J., et al., Radiology 1971, 100, 683-686; Siegle, R. L. and Liebennan, P., Invest Radiology 1976, 11:98-101), the exact mechanism by which this occurred has, until recently, been obscure.
Employing a highly sensitive test for antibody-antigen complexing (passive red blood cell hemagglutination inhibition), we found what we consider the answer to this problem. Ovalbumin and gamma globulin (Sigma) were bound to glutaraldehyde stabilized sheep red blood cells (Inter-Cell Technologies, Hopewell, N.J.). Bis-diazotized benzidine was used to bind the ovalbumin to the RBC's. The gamma-globulin bound directly. In both the case of the ovalbumin and the gamma-globulin, the sensitized red blood cells were allowed to incubate with each of the contrast media (methylglucamine iothalamate [CONRAY; 282 mg iodine/ml, Mallinckrodt Medical, St. Louis Mo.], ioversol [OPTIRAY 320; 320 mg iodine/ml, Mallinckrodt], IOXAGLATE [BEXABRIX; 320 mg iodine/ml, Mallinckrodt] and IOTROLAN [ISOVIST; 300 mg iodine/ml, Schering Pharmaceutical; Berlin, Germany]) and the antibodies at room temperature for 2-3 hours prior to evaluation of the potential of the contrast media to compete with the bound ovalbumin or gamma-globulin for their respective antibodies and thereby to function as “pseudoantigens.” If no competition occurred, there would be a visible agglutination that covered variable areas of the base of the microtiter well, dependent on the strength of the antibody titer. When competition occurred, there would be a diminished diameter of the agglutination, depending on the antibody titer that could be compared to a control, where saline was substituted for the CM. With complete competition (inhibition), no agglutination would be visible and the RBCs would form a small button of cells at the bottom of the microtiter well. Visible agglutination was evaluated on a 1+ to 4+ basis. Studies were also carried out where the RBCs were incubated with CM before or after binding of the antigen to BDB or the RBC to determine whether competition of the CM with the antigen might actually represent displacement of the CM from the RBC surface.
It was discovered that contrast media can in fact act as an antigen and combine with antibodies (Lasser, E. C. and Lanakin, G. E., Academic Radiology 1998, 5 (suppl. 1; S95-S98)). This was shown when, at various concentrations, individual contrast media would inhibit the agglutination of RBC-attached ovalbumin or RBC-attached gamma globulin in the presence of their respective antibodies, thus resulting in a button of cells, rather than agglutination, in the bottom of the microtiter well. Table I shows the results of this experiment.
TABLE Iγ-globulin vs. anti-γ-globulinLowest concentration of CM showing a 2+ or3+ inhibition of 1/500 IgG anti-γ-globulinmg/mlHEXABRIX8.0ISOVIST14.0OPTIRAY16.0Mga LOTHALAMATE28.2Na LOTHALAMATE28.2
The results shown in Table I demonstrate that various contrast media compete successfully for binding to the antibodies awaiting the RBC-antigens and thereby rendering the antibodies unavailable to these antigens. Further studies indicated that this occurred most readily in concentrated CM solutions and that most of the contrast media currently in use tend to aggregate to varying degrees and this was particularly true in more concentrated solutions.
Ovalbumin is known to bind on the variable portion of the immunoglobulin molecule (Fab), while gamma globulin is known to bind to the constant portion of the specific immunoglobulin (Fc) (Frick, O. L. in Basic & Clinical Immunology 2nd Edition, Fudenberg, Stites, Caldwell and Wells editors; Lange Medical Publications; Chapter 22; Immediate Hypersensitivity). Later, the potential of a contrast molecule to compete with ragweed pollen in a ragweed sensitized in vivo rat model (and thereby inhibit the development of ragweed pollen conjunctivitis) was tested. The available data thus far suggests that with local application, the contrast molecule utilized (IODIXANOL; Nycomed, Oslo, Norway) provides a degree of protection by successfully competing with the local application of ragweed antigen and thus inhibiting the potential of the antigen to bind with its specific binding site on anti-ragweed-IgE attached to conjunctival mast cells (see Example V). Thus, it is demonstrated that contrast media have the potential to bind to at least three divergent antibodies and furthermore (inferentially) that binding may take place on either the constant, variable, or both the constant and variable portions of the immunoglobulin molecule.
In the literature, there is a report suggesting that contrast media in vivo reduced the binding of three diverse tumor antigens to their respective antibodies, and thereby falsely lowered the tested blood concentrations of these antigens (Watanabe, N. et al. Nucl. Med. Commun. 1998, 19:63-70). The mechanism for this was not explored in the article, but a careful review of the publication demonstrates that it is likely that the various contrast media were interfering with the ability of the tumor antigens to bind to their respective antibodies.
In view of all of the above information, it is believed that contrast media function as totipotential universal antigens and may thereby compete with any antigen for binding sites on its specific antibody.
Since contrast media, like antigens, can bind to antibodies but cannot themselves produce antibodies (unlike antigens), we have termed the contrast media “pseudoantigens.” It was noted earlier that the contrast media do not have the chemical characteristics to bind to macromolecules and thus do not have attributes to function like classical antigens. The question then arises: how then, do contrast media compete with antigens? In discussing this issue, it is necessary to have information on the general structure of contrast media molecules.
The X-ray contrast media currently available are generally triiodinated, completely substituted, benzene moieties existing in the form of a monomer or a dimer. These contrast media molecules may be either ionic or nonionic (or in the case of one dimer, part ionic and part nonionic). There are generally slight variations in the amide side chains attached at the 3 and 5 positions on the ring and in the nature of the cations (for the ionic media) and there are slight differences in the length of the aliphatic chains linking the dimers and in the nature of the coupler group.
Some examples of X-ray contrast media that are commercially available are METRIZAMIDE, IOPAMIDOL and IOBEXOL which are nonionic monomers. IOXAGLATE and IOTROLAN are ionic dimers. For purposes of this patent application, only nonionic dimers will be considered. The only two ionic dimers believed to be commercially available thus far are IODIXANOL and IOTROLAN. The term “mammal” as used herein refers to human and non-human mammals. Within certain embodiments of the invention, dosage of CM may be from 0.1-40 grams of CM depending on the subject to be treated and the CM. Other dosages of administered CM may be from 0.01-0.1 grams, 0.1-5 grams, 5-10 grams, 10-15 grams, 15-20 grams, 20-25 grams, 25-30 g 30-35 grams, 35-40 grains, 40-45 grams, 45-50 grams and 50-100 grams.
The ability of the contrast media to bind to antibodies must depend on some factor other than their chemical composition since, as noted, their molecular structures do not suggest a potential for binding and in dilute solutions, no binding to globulins could be demonstrated (Lang, J. H. and Lasser, E. C., Invest Radiology 1967, 2:396-400). The explanation appears to be the potential of all of the contrast media, in relatively high concentrations, to aggregate, as determined by both physical-chemical analysis, and by comparing theoretical vs. actual osmolalities (Krause W., et al., Invest Radiology 1994, 29:72-80; Schneider, P., European Radiology 1996, 6:15-16). In an aggregated form, contrast molecules have physical characteristics that simulate a multivalent antigen. In considering the aggregation phenomena it turns out, counter-intuitively, that the best aggregators, and the best antibody binders, are also the contrast media least likely to produce adverse reactions on injection into animals or humans. Under normal circumstances one would expect that the molecule most likely to promote antibody-antigen reactions would be the molecule most likely to play a role in adverse reactions.
In attempting to solve this paradox, a study done much earlier in our laboratory is referenced, wherein dogs injected with a constant volume of contrast media over either a 2 second or 10 second interval consistently produced a higher concentration of histamine release with the longer interval (Lasser, E. C. et al., Radiology 1971, 100:683-686).
Histamine release from mast cells and basophils is known to occur when adjacent IgE antibodies attached to these cells are connected by a bridging antigen. Under these circumstances, the receptors that bind the antibodies to the cells are believed to be activated to induce phospholipid methylation and an increase in intracellular cyclic AMP. These biochemical events are followed by an influx of calcium and the release of histamine (Ishizaka, T. et al., J Immunology 1983, 130:2357-62). Given these facts, it appeared paradoxical that the faster (2 sec.) injection which should have presented the antibodies on the cells with a higher concentration of contrast media and thus a higher concentration of “pseudoantigens” (and hence greater histamine release) actually resulted in less histamine release than the slower injection.
Further analysis of this paradox pointed to the phenomenon of “antigen-excess.” Antigen-excess in vitro has been recognized for many years (Myrvik, Q. N. and Weiser, R. S.—Fundamentals of Immunology, Second Edition: Lea and Febiger, Philadelphia 1984, 96102). When a sufficiently concentrated antigen is added to a solution of its specific antibody, there will be successive phases of antibody-excess, antibody-antigen equivalence and finally antigen-excess. In most cases at antibody-antigen equivalence, a precipitate will develop. In antigen-excess, soluble compounds (antigen-antibody complexes) will remain in solution in the supernatant so that precipitation is less than maximal. With a large excess of antigen, inhibition of precipitation may become complete. FIG. 3 depicts our interpretation of the antigen (“pseudoantigen”)-excess phenomenon as it applies to CM binding to IgE immunoglobulins on mast cells.